The first broad, long-term objective of this research is to obtain quantitatively accurate descriptions of the molecular structure and organization of biomembranes. This includes structure in the direction along the bilayer normal, such as thickness and'electron density profiles. It also includes structure in the plane of heterogeneous membranes such as sphingomyelin:DOPC:cholesterol, in order to evaluate current concepts of rafts. Our focus is on small scale heterogeneity, which is indicated by many studies on biomembranes, rather than on large scale thermodynamic phase coexistence. We will test the hypothesis that small scale heterogeneity robustly exists in model membrane systems. Our structural studies include studying the effect of peptides on both homogeneous lipid bilayers and on laterally heterogeneous mixtures, to investigate where peptides reside in the bilayer, and the effect of cholesterol sequestering peptides, such as N-acetyl-LWYDC- amide, on raft formation. Another focus will be on the HIV-1 fusion peptide FP-23 that plays a crucial role in initiating AIDS infection. We will test the hypothesis that insertion geometry depends upon the lipid composition of the target membrane and upon oligomerization of the FP-23 peptide. Our structural studies include studying the effect of ion channel modulators on membranes. Our focus is on genistein which modulates the cystic fibrosis transmembrane regulator (CFTR) channel. We will test the hypothesis that genistein affects the membrane thickness and stiffness, which then modulates channel characteristics. The second broad objective is to determine quantitatively the interactions between membranes and their mechanical properties at the nanoscale. Our focus is on the entropic fluctuation force and on the bending modulus and how these depend upon membrane composition. This second objective will provide data, such as the bending modulus and the strength of the van der Waals interactions, that are fundamental in biophysical modeling of many biological processes. The phenomenological physical link between these 2 broad objectives is the essential role that fluctuations play in biomembranes. Diffraction methodology that was developed in the previous grant period takes these fluctuations into account and uses them to advantage. Our primary and innovative technique is the measurement and interpretation of x-ray diffuse scattering in oriented samples from which data can be obtained to higher q even for frilly hydrated samples, thereby providing better structure in more biologically relevant samples. This will be complemented by newly accessible neutron scattering experiments, as well as by our volumetric measurements and Monte Carlo simulations.